Pancreatic Transdifferentiation and Glucose-Regulated Production of Human Insulin in the H4IIE Rat Liver Cell Line

Abstract

Due to the limitations of current treatment regimes, gene therapy is a promising strategy being explored to correct blood glucose concentrations in diabetic patients. In the current study, we used a retroviral vector to deliver either the human insulin gene alone, the rat NeuroD1 gene alone, or the human insulin gene and rat NeuroD1 genes together, to the rat liver cell line, H4IIE, to determine if storage of insulin and pancreatic transdifferentiation occurred. Stable clones were selected and expanded into cell lines: H4IIEins (insulin gene alone), H4IIE/ND (NeuroD1 gene alone), and H4IIEins/ND (insulin and NeuroD1 genes). The H4IIEins cells did not store insulin; however, H4IIE/ND and H4IIEins/ND cells stored 65.5 ± 5.6 and 1475.4 ± 171.8 pmol/insulin/5 × 10⁶ cells, respectively. Additionally, several β cell transcription factors and pancreatic hormones were expressed in both H4IIE/ND and H4IIEins/ND cells. Electron microscopy revealed insulin storage vesicles in the H4IIE/ND and H4IIEins/ND cell lines. Regulated secretion of insulin to glucose (0-20 mmol/L) was seen in the H4IIEins/ND cell line. The H4IIEins/ND cells were transplanted into diabetic immunoincompetent mice, resulting in normalization of blood glucose. This data shows that the expression of NeuroD1 and insulin in liver cells may be a useful strategy for inducing islet neogenesis and reversing diabetes.

Keywords: H4IIE cells; diabetes; furin-cleavable human insulin; gene therapy; insulin storage; liver cells; regulated insulin secretion; secretory granules; β cell transcription factors.

Figure 1

Insulin secretion from H4IIE/ND and H4IIEins/ND cells: (A) dose response curve to increasing concentrations of glucose, 0–20 mmol/L; (B) response to 10 mmol/L theophylline; and (C) 10 mmol/L calcium. Cells were incubated in the basal medium for two consecutive 1 h periods (Basal 1 and 2) before exposure to the stimulus for 1 h, followed by a third basal period (Basal 3). Values are expressed as means ± SE (n = 6).

Figure 2

Transmission electron micrographs of: (A) H4IIEins/ND cells showing secretory granules (SG) surrounded by a pale halo; (B) H4IIE/ND cells showing immature secretory granules (marked with black arrows) and one larger secretory granule with a pale halo (marked by white arrow). The insert shows an immunoelectron micrograph (IEM) of a granule labelled with 10 nm gold particles; (C) IEM of H4IIEins/ND cells; and (D) MIN6 cells showing secretory granules labelled with 10 nm gold particles to show where insulin was stored in the cells (selected granules marked with arrows).

Figure 2

Transmission electron micrographs of: (A) H4IIEins/ND cells showing secretory granules (SG) surrounded by a pale halo; (B) H4IIE/ND cells showing immature secretory granules (marked with black arrows) and one larger secretory granule with a pale halo (marked by white arrow). The insert shows an immunoelectron micrograph (IEM) of a granule labelled with 10 nm gold particles; (C) IEM of H4IIEins/ND cells; and (D) MIN6 cells showing secretory granules labelled with 10 nm gold particles to show where insulin was stored in the cells (selected granules marked with arrows).

Figure 3

Reversal of diabetes in NOD/scid mice after transplantation of H4IIEins/ND cells: (A) blood glucose levels of NOD/scid mice following transplantation with H4IIEins/ND cells, together with diabetic controls. Grafts of H4IIEins/ND cells were removed at 24 days; and (B) intraperitoneal glucose tolerance test in mice following transplantation with H4IIEins/ND cells, together with normal (non-diabetic) and diabetic controls. Values are expressed as mean ± SEM (n = 7).

Figure 4

Expression of pancreatic hormones and NEUROD1 in grafts from NOD/scid mice following transplantation of H4IIEins/ND cells and diabetes reversal. Photomicrographs of double-stained anti-insulin and anti-NEUROD1 of (A) H4IIEins/ND cells; and (B) tissue near graft (bar = 80 µm); (C–E): Photomicrographs of triple anti-insulin (INS), anti-glucagon (GLUC) and anti-somatostatin (SST) staining of (C) H4IIEins/ND graft; (D) normal mouse pancreas; and (E) diabetic mouse pancreas (bar = 80 µm). Original magnification 200×.

Figure 5

Pancreatic hormones and β cell transcription factors expressed in rat liver cell lines. (A) reverse transcription polymerase chain reaction (RT-PCR)analysis of cell lines for INS-FUR, NeuroD1 and β-actin (Actb): H4IIE (lane 1), H4IIEins (lane 2), H4IIE/ND (lane 3), and H4IIEins/ND (lane 4); (B) RT-PCR analysis for: β cell transcription factors (Pdx1, Neurog3, NeuroD1, Nkx2-2, Nkx6-1, Pax6, MAFA, MAFB); the rat pancreatic endocrine hormones insulin 1 and 2 (Ins1, Ins2), glucagon (Gcg), somatostatin (Sst), and pancreatic polypeptide (Ppy); GLUT2 (Slc2a2) and glucokinase (Gck); insulin proconvertase PC1 (PC1) and PC2 (PC2); the exocrine marker p48; the potassium channel proteins SUR 2A, SUR2B, Kir 6.1 and Kir 6.2 and β-actin in H4IIE (lane 1), H4IIEins (lane 2), H4IIE/ND (lane 3), H4IIEins/ND (lane 4), and rat pancreatic tissue (lane 5).